The development of axon pathways is a complex signal transduction process linking extracellular guidance cues to intracellular signals regulating axon outgrowth and steering. Guidance errors lead to structural birth defects in the nervous system that cause physical, mental, or behavioral impairment. The long-term goal of the investigator's laboratory is to understand how signaling events lead to the development of axon pathways and functional connectivity. Drosophila will be used as the model system as it allows molecular, genetic, and cellular tools to be applied to the in vivo study of axon guidance, while gene conservation makes the study relevant to human development. The goal of this application is to establish a role for G-protein coupled receptors (GPCRs) in axon pathway formation during development of the Drosophila embryonic nervous system. GPCRs are transmembrane receptors that activate trimeric G-proteins to regulate several key intracellular signaling pathways. GPCRs may directly function as guidance cue receptors, or modify second messenger levels to indirectly affect the signaling potential of other ligand-receptor systems operating at a guidance choice point. Based on vertebrate and Drosophila studies, it is hypothesized that GPCRs function in the developing Drosophila nerve cord to guide axons to their targets. Specific Aim 1 will test this hypothesis by systematically evaluate the role of GPCRs in axon pathway formation in the Drosophila embryo by genetically altering components of the canonical GPCR signaling pathway;and Specific Aim 2 will identify GPCRs that guide the development of axon pathways. Aim 1 tests how mutations in various genes function in GPCR signaling pathways alter axon pathway formation alone or in combination with other guidance molecules. It documents the importance of GPCR signaling to pathway formation and addresses how they do so. Aim 2 uses standard RNA hybridization methods to identify GPCRs expressed in the nerve cord. New preliminary data have led the investigator to focus on Methuselah and its Methuselah-like cousins, two of which are expressed in the nerve cord during axonogenesis. Mutations in these genes are used to assess the importance of expression to axon guidance. Future R01 applications will harness the Drosophila "toolbox" to delineate the molecular mechanism by which a GPCR participates in axon guidance decisions. Understanding the fundamental processes underlying axon pathway formation, and their association with structural birth defects, is a critical component of the NIH mission. Elucidating the mechanisms of GPCR activity also contributes to our understanding of several neurological and behavioral pathologies. PROJECT NARRATVE: Errors in the development of axon pathways lead to structural birth defects, which in turn lead to physical, mental, and behavioral impairments significantly impacting a person's lifestyle and the national economy. This application uses the development of the Drosophila embryo as a model system to understand the fundamental processes underlying development of axon pathways in the spinal cord. Understanding these processes is an important step in devising therapeutic interventions and improving the quality of life of affected individuals.